CN117305187A - Pediococcus acidilactici for improving intestinal health condition and application thereof - Google Patents

Pediococcus acidilactici for improving intestinal health condition and application thereof Download PDF

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CN117305187A
CN117305187A CN202311576192.6A CN202311576192A CN117305187A CN 117305187 A CN117305187 A CN 117305187A CN 202311576192 A CN202311576192 A CN 202311576192A CN 117305187 A CN117305187 A CN 117305187A
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pediococcus acidilactici
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intestinal
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张旭朏
李璟欣
李鑫
王梦创
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Sichuan Anaerobic Biotechnology Co ltd
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Abstract

The invention belongs to the field of microorganisms, and particularly discloses pediococcus acidilactici for improving intestinal health conditions and application thereof. The pediococcus acidilactici Pacid-1 has no virulence factor, is not hemolytic, is sensitive to various antibiotics, and has good safety. The strain has good tolerance to artificial gastric juice, can inhibit various pathogenic bacteria, relieve barrier dysfunction, inhibit the expression of proinflammatory factors, and can effectively improve gastrointestinal symptoms.

Description

Pediococcus acidilactici for improving intestinal health condition and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to pediococcus acidilactici for improving intestinal health conditions and application thereof.
Background
Intestinal microorganisms are closely related to human health and are visually known as "microbial organs". The intestinal flora is used as an important component of the organism, keeps dynamic and stable under normal conditions, and plays an important role in promoting digestion and absorption of nutrient substances, maintaining normal physiological functions of the intestinal tract, regulating various vital activities of the organism, such as immunity and the like. However, the intestinal flora is susceptible to various factors, such as: environmental factors, eating and living habits, mental factors, disease state, tumor treatment, antibiotic use, and age.
The human intestinal flora is disturbed after being influenced by the factors, namely the disturbance of the intestinal flora, which can be manifested by the deficiency of beneficial intestinal bacteria, the excessive reproduction of pathogenic bacteria, the damage of intestinal barrier function and the occurrence of intestinal inflammation, and further causes gastrointestinal diseases of a host, such as constipation, diarrhea, abdominal pain, abdominal distention and the like, and can be seriously developed into diseases such as inflammatory bowel disease, ulcerative colitis, irritable bowel syndrome and the like, thereby greatly affecting the health and life quality of the human body.
Currently, there is increasing interest in improving intestinal health and preventing or treating intestinal diseases using intestinal probiotics. The intestinal probiotics can strengthen the barrier function of intestinal mucosa, prevent adhesion and colonization of pathogenic bacteria and strengthen immune response of a system, thereby achieving the effect of maintaining intestinal health. For example, chinese patent application CN 102711778A discloses a bifidobacterium animalis subspecies lactis DN-173010 and has been verified by mouse experiments and histological studies that fermented milk thereof can alleviate ulcerative colitis. Patent application publication No. CN107312726A discloses a Lactobacillus plantarum which inhibits the growth of harmful bacteria in the intestine, such as E.coli, salmonella, streptococcus suis, and Staphylococcus aureus.
Probiotics are also used to prevent or ameliorate side effects caused by some drugs, such as antibiotics. Side effects associated with chemotherapy are also common in clinic. Digestive system reactions are one of the most common side effects such as nausea, vomiting, diarrhea, constipation, etc. Taking 5-FU as an example, after 5-FU is phosphorylated to 5-FdUMP or 5-FUMP, the 5-FU is more sensitive to the proliferated small intestinal cells, and can cause damage to the small intestinal mucosa and interfere with division of the intestinal cells to cause necrosis of intestinal wall cells and extensive inflammation of intestinal wall, so that unbalance of the number of absorbed and secreted cells is caused, and diarrhea is caused. In addition, chemotherapeutics can also cause cellular DNA damage and mitochondrial dysfunction, leading to ROS production and apoptosis. ROS can induce NF- κb activation, further up-regulating the expression of pro-inflammatory factors, leading to damage of epithelium, endothelium and connective tissue. Under the condition that intestinal epithelium is damaged, harmful bacteria are very easy to colonize, intestinal microecology is destroyed, pathogenic bacteria are further caused to infect, and diarrhea is promoted to develop.
Pediococcus acidilactici is one of the important members of the intestinal probiotics. Wentao Li et alMicroorganisms. 2022, 10(12):2350.) The research shows that the Pediococcus acidilactici has better improvement effect on the intestinal inflammation of mice induced by ETEC (enterotoxigenic escherichia coli). In addition, patent CN113736695B discloses that pediococcus acidilactici probiotic strains can improve the symptoms of colitis by improving colonic mucosal injury, inhibiting the expression of pro-inflammatory factors, reducing endotoxin and the like. However, there has been no study reporting the effect of Pediococcus acidilactici on diarrhea caused by chemotherapeutic drugs.
Disclosure of Invention
The invention firstly provides a Pediococcus acidilacticiPediococcus acidilactici) The strain is selected from Pediococcus acidilactici Pacid-1 with the preservation number of CCTCC NO: M20222030 or a passage strain with the toxicity, immunogenicity and biological activity unchanged from those of Pediococcus acidilactici Pacid-1 with the preservation number of CCTCC NO: M20222030.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) The 16S rDNA sequence of (2) is identical with SEQ ID NO. 1.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the gene of coding the enzyme related to the acetic acid with the amino acid sequence shown as SEQ ID NO. 2.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the gene of coding the enzyme related to the acetic acid with the amino acid sequence shown as SEQ ID NO. 3.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the gene of coding the enzyme related to the acetic acid with the amino acid sequence shown as SEQ ID NO. 4.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the gene of coding the enzyme related to the acetic acid with the amino acid sequence shown as SEQ ID NO. 5.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the gene for coding the enzyme related to the propionic acid with the amino acid sequence shown as SEQ ID NO. 6.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the gene for encoding CAT (catalase) related enzyme with the amino acid sequence shown in SEQ ID NO. 7.
Secondly, the invention also provides the Pediococcus acidilacticiPediococcus acidilactici) The method for culturing the strain comprises the steps of inoculating the pediococcus acidilactici strain into a culture medium, and performing proliferation culture to obtain the proliferated pediococcus acidilactici strain.
In some embodiments, the medium contains 15-20 g of BHI broth powder, 10-15 g of MRS broth powder, and 12-17 g of modified GAM broth powder per 1L distilled water.
The invention also provides a composition, the active ingredient of which contains the pediococcus acidilacticiPediococcus acidilactici) The strain or the strain containing Pediococcus acidilactici obtained by the culture methodPediococcus acidilactici) Strains.
In some specific embodiments, the pediococcus acidilacticiPediococcus acidilactici) The strain was used as the sole active ingredient.
Finally, the invention alsoProvides the Pediococcus acidilacticiPediococcus acidilactici) Or the application of the composition in preparing products for improving intestinal health.
In some embodiments, the intestinal health condition is intestinal inflammation and/or intestinal barrier damage and/or intestinal pathogen infection.
In some embodiments, the intestinal health condition is diarrhea caused by a chemotherapeutic agent.
In some embodiments, the enteropathogenic bacteria are selected from any one or a combination of the following: pseudomonas aeruginosa, shigella dysenteriae, yersinia enterocolitica and Clostridium difficile.
In some embodiments, the chemotherapeutic agent is selected from one or a combination of the following: 5-fluorouracil, tegafur, 5'-2' -deoxyuridine, capecitabine, tegafur, paclitaxel, docetaxel, vinorelbine, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, ifosfamide, melphalan, carmustine, irinotecan.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the following biochemical identification characteristics:
1) Colony morphology on anaerobic triple mixed culture medium (BHI+MRS+modified GAM) is white opaque round, middle bulge and smooth and moist surface;
2) Genome avirulence gene;
3) In vitro non-hemolysis (i.e., gamma hemolysis);
4) Sensitive to at least two antibiotics selected from penicillin, ampicillin, imipenem, ceftriaxone, tetracycline, clindamycin, levofloxacin, nitrofurantoin, high concentration gentamicin, streptomycin, rifampin;
5) Is resistant to artificial gastric juice.
In some specific embodiments, the pediococcus acidilactici of the inventionPediococcus acidilactici) Has the following function authentication characteristics:
1) Has intestinal barrier repairing function;
2) Has in vitro anti-inflammatory effect;
3) Can prevent, improve, alleviate or relieve diarrhea associated with chemotherapy;
4) Has antibacterial activity against Pseudomonas aeruginosa, shigella dysenteriae, yersinia enterocolitica and Clostridium difficile.
The pediococcus acidilactici has the following characteristics:
1. no toxicity factor, no hemolysis, sensitivity to various antibiotics and good safety;
2. can resist artificial gastric juice, has inhibiting effect on various pathogenic bacteria, can improve intestinal barrier, inhibit the expression of proinflammatory factors, and can prevent, improve, alleviate or relieve diarrhea caused by chemotherapy drugs;
3. the effect of improving diarrhea caused by the chemotherapeutic medicine is equivalent to that of the chemical agent loperamide.
The strain preservation information of the invention is as follows:
strain name: pediococcus acidilacticiPediococcus acidilactici）Pacid-1
Preservation date: 2022 12/23
Preservation unit: china center for type culture collection (China Center for Type Culture Collection, CCTCC), address: university of martial arts, hubei province, post code: 430072, telephone: 027-68754052.
Preservation number: cctccc No. M20222030.
Drawings
FIG. 1 is a front photograph of the morphology of Pediococcus acidilactici Pacid-1 colonies of example 1.
FIG. 2 is a graph showing the results of Pediococcus acidilactici Pacid-1 artificial gastric juice resistance of example 5.
FIG. 3 is a graph showing the results of an antibacterial test of Pediococcus acidilactici Pacid-1 of example 6.
FIG. 4 shows the results of the Pediococcus acidilactici Pacid-1 pair barrier repair test of example 7.
FIG. 5 is a graph showing the results of an experiment for inhibiting inflammatory expression of cells by Pediococcus acidilactici Pacid-1 of example 8 (A. Sup. Ply of IL-6 expression; B. Sup. Ply of TNF-. Alpha. Expression).
FIG. 6 is a graph showing the effect of Pediococcus acidilactici Pacid-1 treatment on 5-fluorouracil diarrhea mice of example 9 (day 1-9 diarrhea score curves for each group A; B D diarrhea score graph; C diarrhea total score graph for each group).
Detailed Description
Definition and description
For the strain claimed in the present invention (Pediococcus acidilactici strain with a microorganism accession number of CCTCC NO: M20222030, pacid-1 strain), passaging strains which are identical to the genome of Pacid-1 strain without mutation or accumulate minute mutations in passaging, but have NO substantial changes in toxicity, immunogenicity and biological activity should be regarded as Pacid-1 strains deposited with microorganisms. The passaged strain or mutant strain having no substantial change in toxicity, immunogenicity and biological activity mainly refers to a strain based on Pacid-1 strain passaged and accumulated with minute mutations in passaging. And the strain includes live bacteria and inactivated forms, whole thalli or lysate thereof or fermentation products thereof.
The propagation application of Pacid-1 strain inevitably introduces tiny mutation, and the passage strain or mutant strain with no substantial change of toxicity, immunogenicity and biological activity is considered to be within the contribution scope of the invention. There is no substantial change in toxicity, immunogenicity, and biological activity, including, but not limited to, regarding toxicity, immunogenicity, and biological activity as being the same within the limitations and acceptable or unavoidable errors of detection techniques such as detection sensitivity, detection limits, and the like.
It is often desirable to determine toxicity, immunogenicity, and biological activity of the Pacid-1 offspring from animals, and it is desirable that the offspring be of passaged strains that have no substantial changes in toxicity, immunogenicity, and biological activity due to differences in animal species, age, sex, health, etc., and that systematic errors that can be expected or unavoidable.
It is inevitable to introduce minute mutations after passage of Pacid-1 strain a plurality of times, and it is reasonable to expect that these minute mutations remain within the range of the essential technical contribution of the present invention, when they occur in non-coding sequence regions or synonymous mutations of coding regions or mutations that do not affect strain toxicity, immunogenicity and biological activity (for example, residues that may be linked amino acid residues between two domains or residues that are located within the higher structure of a protein and do not affect toxicity, immunogenicity and biological activity by not contacting immune cells), these minute mutations still belong to insubstantial mutations, and should be regarded as mutant strains that have no change in toxicity, immunogenicity and biological activity.
The culture medium of Pacid-1 strain of the present invention cultures passaged strains, and it is reasonable to expect that, like other bacteria, it is inevitable to introduce minute mutations, which are passaged strains or mutant strains having no substantial changes in toxicity, immunogenicity and biological activity when they have no substantial changes in toxicity, immunogenicity and biological activity.
Pacid-1 strains are derived from human feces, and it is necessarily possible to isolate and identify homologous strains in different humans or in the environment, which have a common ancestor with Pacid-1 strains and have obvious physiological genetic differences with other known Pediococcus acidilactici strains, and their genomes may be identical to that of Pacid-1 strains, or may have minor differences.
When these homologous strains differ from Pacid-1 strain to the extent corresponding to the extent of the differences between the strain of passaging or mutant strain, which have no substantial changes in toxicity, immunogenicity and biological activity, and Pacid-1 strain, these homologous strains are identical to Pacid-1 strain or are considered to have no differences in toxicity, immunogenicity and biological activity, and these homologous strains belong to the strain substantially identical to Pacid-1 strain.
The composition contains an active ingredient pediococcus acidilactici and other ingredients, such as auxiliary ingredients with no physiological effects or other functional ingredients. The functional components include, but are not limited to, other functional strains, or nutritional components with nutritional, dietary supplement, dietary fibers, prebiotic components, metagen components, and the like.
The composition of the present invention may be prepared in any form convenient for use, such as powder, tablet, granule, gel, capsule or liquid, which are common in clinical or food.
The compositions of the present invention are administered to a subject in an amount (therapeutically effective amount) and frequency effective to exert efficacy, preferably in a single dose of 10 2 ~10 15 CFU、10 4 ~10 13 CFU or 10 5 ~10 12 Pediococcus acidilactici of CFUPediococcus acidilactici）。
The diarrhea refers to a clinical symptom that the defecation frequency is obviously higher than that of usual habits (> 3 times/d), the feces are thin, the water content is increased (> 85%), and the feces are accompanied with mucus, sepsis or undigested food.
The intestinal inflammation refers to intestinal inflammatory reaction caused by various reasons such as microbial infection, ischemia, radioactive rays, organism immunity disorder and the like, and the most common symptoms are abdominal pain, diarrhea, bloody stool, fever and the like. Can be accompanied with the increase of inflammatory factor indexes such as interleukin-6 (IL-6).
The intestinal barrier damage refers to the damage of the barrier function of intestinal mucosa, which leads to the change of intestinal permeability, and intestinal endotoxin, bacteria and other harmful substances enter the blood circulation system to cause a series of inflammatory reactions and related diseases. The increased intestinal permeability may reflect damage to the intestinal mucosa and is an important indicator for evaluating the intestinal barrier function.
The specific temperature parameters in the present invention, unless specified otherwise, are understood to be constant temperature treatments and allow for variations within a certain temperature interval. Such as within a range of + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.
The auxiliary materials comprise a drug carrier and an excipient. A pharmaceutical carrier refers to a pharmaceutical carrier that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered probiotic. The pharmaceutically acceptable carrier may enhance or stabilize the composition or may be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers can include solvents, dispersion media, coatings, surfactants, antioxidants, isotonic agents, absorption delaying agents, salts, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., remington's Pharmaceutical Sciences, 18 th edition MackPrinting Company,1990, pages 1289-1329). Unless the conventional carrier is incompatible with the active ingredient, it is contemplated that it will be used in a therapeutic or pharmaceutical composition. The carrier may be selected to minimize adverse side effects in the subject and/or minimize inactivation of the active ingredient.
An excipient refers to a substance that is added to a pharmaceutical composition to give the drug a certain shape or a certain concentration. Such as sterile water, physiological saline, polyalkylene glycols (such as polyethylene glycol), vegetable oils or hydrogenated naphthalenes, calcium bicarbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, and the like.
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by a person skilled in the art without making creative efforts based on the embodiments in the present invention shall fall within the protection scope of the present invention.
The preparation method of the culture medium used in the following examples is as follows:
preparation of YCFA liquid culture medium: casein 10.0. 10.0 g, yeast extract 2.5 g, mgSO were weighed out 4 ·7H 2 O0.45 mL (10% mother liquor), 10 mg/mL CaCl 2 Solution 0.45 mL,TE141 10 mL,K 2 HPO 4 0.45 g，KH 2 PO 4 0.45 g, naCl 0.90 and g are dissolved in a proper amount of distilled water, and the heating is stopped after the solution is heated and boiled. In the cooling process of the culture medium, naOH is added into the VFA-mix of 3.2 mL in batches to adjust the pH value to be neutral, the culture medium is added into the culture medium after being cooled to the room temperature, then adding 0.5 g of cysteine hydrochloride monohydrate, uniformly stirring 0.1% of resazurin 1 mL, regulating pH to neutral by NaOH, heating again for boiling, maintaining micro-boiling state for about 20 min, and stopping heating, N 2 Replacing, cooling, packaging, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and dry place.
Preparation of TE 141: adding nitrilotriacetic acid 1.50. 1.50 g into 200. 200 mL pure waterAdding appropriate amount of NaOH until the solution becomes clear, adding 800-mL water, adjusting pH to 5.5 with 50% HCl, and sequentially weighing MgSO 4 ·7H 2 O 3.00 g，MnSO 4 ·H 2 O 0.50 g，NaCl 1.00 g，FeSO 4 ·7H 2 O 0.10 g，CoSO 4 ·7H 2 O 0.18 g，CaCl 2 ·2H 2 O 0.10 g，ZnSO 4 ·7H 2 O 0.18 g，CuSO 4 ·5H 2 O 0.006 g，KAl(SO 4 ) 2 ·12H 2 O 0.02 g，H 3 BO 3 0.01 g，Na 2 MoO 4 ·2H 2 O 0.01 g，NiCl 2 ·6H 2 O0.03 g,10 mg/mL Na 2 SeO 3 ·5H 2 O solution 0.03 mL,10 mg/mL Na 2 WO 4 ·2H 2 And adding the O solution 0.03. 0.03 mL into the test solution, and continuously stirring in the adding process to keep the solution clear for later use.
Preparation of VFA-mix: taking 90 mL parts of acetic acid, 30 mL parts of propionic acid, 10 mL parts of n-valeric acid, 10 mL parts of isobutyric acid and 10 mL parts of butyric acid, mixing for later use, and adjusting the pH to be neutral by using a 5M concentration NaOH solution before use.
Preparation of original sample protectant: weighing Na 2 HPO 4 ·12H 2 O 3.85g，KH 2 PO 4 0.27 g, naCl 8.00 and g, adding a proper amount of distilled water for fully dissolving, adding 200 mL glycerol, supplementing water to 1.2 and L, heating and boiling, and introducing N 2 Cooled to room temperature. Adding 1.00 g cysteine salt and 0.1% of resazurin 1 mL, dissolving, adjusting pH to neutral with 5M NaOH solution, boiling again until the color of the protectant is colorless, heating for more than 20 min, and introducing N 2 Cooling to room temperature, packaging, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and dry place.
Preparation of triple mixed media (bhi+mrs+modified GAM): weighing 19.25 g of BHI broth powder (Qingdao sea Bo Biotechnology Co., ltd., HB 8297-5), 13.5 g of MRS broth powder (Guangdong CycloKai Biotechnology Co., ltd., 027312), 15 g of modified GAM broth powder (Qingdao sea Bo Biotechnology Co., ltd., HB 8518-3) and adding agar powder 12 g when preparing a triple-mixed solid culture mediumDissolving in distilled water of 1L, N 2 Removing oxygen, packaging, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and dry place.
Preparation of a two-mixed culture medium (bhi+mrs): weighing 19.25 g of BHI broth powder, 27.0. 27.0 g of MRS broth powder, 0.5. 0.5 g of cysteine hydrochloride monohydrate (Emeishan dragon biotechnology Co., ltd.) and dissolving in 1. 1L of distilled water, deoxidizing, packaging, sterilizing at 121deg.C under moist heat for 15 min, and storing in shade and dry place. Preparation of MRS broth: weighing MRS broth powder 54.0 g, cysteine hydrochloride monohydrate (Emeishan mountain Dragon Biotechnology Co., ltd.) 0.5. 0.5 g, dissolving in distilled water of 1L, N 2 Replacement deoxidization, and sterilization at 121 ℃ for 15 min.
The preparation of MRS solid culture medium, GAM solid culture medium, TSB (tryptone soybean broth, qingdao sea Bo Biotechnology Co., ltd., HB 4114), TSA (tryptone soybean agar, qingdao sea Bo Biotechnology Co., ltd., HB 4138) and Broth (Qingdao sea Bo Biotechnology Co., ltd., HB 0241) culture medium was weighed and dissolved according to the instructions, and the culture medium was sterilized by heat and humidity at 121℃for 30 min and stored in a cool and dry place.
Preparing artificial gastric juice: pepsin (Shanghai derived leaf Biotechnology Co., ltd., S10028) was supplemented in pH3.0 artificial gastric juice (enzyme-free, sterile, shanghai derived leaf Biotechnology Co., ltd., R24026) to a final concentration of 10 g/L, and N was continuously fed in 2 1 h. The solution was sterilized in a glove box with a 0.22 μm filter and stored at 4℃for no more than 30 d.
Preparing a bacterial powder preparation culture medium: weighing glucose 12. 12 g, soybean peptone 5g, yeast extract 5g, yeast peptone 5g, sodium acetate 5g, potassium dihydrogen phosphate 4.5. 4.5 g, disodium hydrogen phosphate dodecahydrate 4.7. 4.7 g, magnesium sulfate 0.1 g, manganese sulfate 0.045 g, tween 80 1 g, cysteine hydrochloride monohydrate 0.5 g, dissolving in distilled water of 1L, N 2 Removing oxygen by replacement, packaging, and sterilizing at 121deg.C for 15 min. Stored in a cool and dry place.
EXAMPLE 1 isolation, identification and preservation of Pediococcus acidilactici Pacid-1
Pediococcus acidilactici（Pediococcus acidilactici) The screening and separating method of Pacid-1 is as follows:
(1) Collecting fresh fecal samples of healthy volunteers, weighing 1-2 g of samples in a 50 mL centrifuge tube, adding an original sample protecting agent according to the mass-to-volume ratio of 1:10, and fully vibrating to resuspend the samples. At N 2 Under the protection, the warp cloth filters the re-suspension bacteria liquid into a 50 mL centrifuge tube, and then the re-suspension bacteria liquid is transferred into a glove box for sub-packaging. During sub-packaging, a 2 mL screw cap tube is used, each sub-packaging is 1 mL, after sub-packaging, labels are attached, the sub-packaging is carried out in a bagging mode, and vacuum pumping is carried out, and the sub-packaging is stored in a refrigerator at the temperature of minus 80 ℃ for standby.
(2) During separation, 1 frozen sample tube is taken and transferred into a glove box for thawing. After the sample is thawed, a pipettor is used for taking 0.5 mL bacterial suspension, and the suspension is uniformly mixed in 4.5 mL anaerobic PBS in an oscillating way, and the suspension is diluted to 10 in a gradient way -6 Mixing proper gradient bacteria liquid and YCFA culture medium, distributing to 384-well plate, and anaerobic culturing at 37deg.C for one week. OD monitoring is provided, grown hole site bacterial liquid is selected and transferred into a new 384-well plate, after the culture is carried out in duplicate, 48 h is cultivated, one part is detected by using MALDI-TOF-MS, the separated bacterial strains are initially classified, the other part is transferred into a 96-well plate again according to mass spectrum results, after the culture is carried out in duplicate, 48 h is cultivated, 16S rDNA gene amplification is carried out on one plate, and sequencing is carried out by the division of Beijing engine biotechnology company, and the other plate is used for sequencing according to 1:1 adding 50% glycerol, uniformly mixing, temporarily preserving, and using after confirming the PCR result.
(3) Analysis of the 16S rDNA gene sequencing results, comparison of the sequence with NCBI nucleic acid database, and the result shows that the sequence is identical to that of Pediococcus acidilacticiPediococcus acidilactici) Sequence similarity of the highest degree>99%) of the isolated strain was initially identified as Pediococcus acidilacticiPediococcus acidilactici) Named Pediococcus acidilactici Pacid-1. After the culture of the three-mixed culture medium, the colony form is white opaque circular colony, the middle is convex, the surface is smooth and moist, and the front photograph is shown in figure 1.
The same screening separation method is adopted to separate and obtain a pediococcus acidilactici strainPediococcus acidilactici) Pacid-2 was used as a control strain for example 9 below.
EXAMPLE 2 Whole genome analysis of Pediococcus acidilactici Pacid-1
Pediococcus acidilactici Pacid-1 is inoculated into 5 mL anaerobic triple mixed culture medium according to the inoculation amount of 2 percent, the culture is carried out until the late logarithmic growth phase, the whole genome DNA of the strain is extracted, and the genome sequencing is carried out by utilizing an Illumina high-throughput sequencing platform Novaseq 6000. After assembly and annotation, the protein sequences were entered into virulence gene bank Virulence Factor Databases (VFDB) for virulence factor analysis. The results show that the bacteria do not have virulence factors.
The novel analysis of the strain was performed using the average nucleotide similarity (Average Nucleotide Identity, ANI). By searching in Genbank, 503 published results were foundPediococcus acidilacticiThe whole genome, through fastANI (v 1.33) comparison, shows that two strains closest to the whole genome of Pediococcus acidilactici Pacid-1 are GCA_009913875.1 (ANI=99.93%) and GCA_015557285.1 (ANI=99.92%) respectively, and are lower than 99.99%, so that Pediococcus acidilactici Pacid-1 can be considered as a new strain, and the 16S rDNA sequence of the new strain is shown as SEQ ID NO. 1.
Annotation of the whole genome sequence by means of emereper-2.1.9 further revealed that Pediococcus acidilactici Pacid-1 can encode 4 acetogenic related enzymes having the sequences shown in SEQ ID NO:2,SEQ ID NO:3,SEQ ID NO:4 and SEQ ID NO:5, 1 propionic acid-producing related enzyme having the sequence shown in SEQ ID NO:6, and 1 CAT (catalase) -producing related enzyme having the sequence shown in SEQ ID NO:7, respectively.
The strain is preserved in China center for type culture collection (CCTCC, university of Chinese, etc.) at 12 months and 23 days 2022, and the preservation number of the strain is: cctccc No. M20222030.
EXAMPLE 3 Pediococcus acidilactici Pacid-1 hemolysis assay
The preserved Pediococcus acidilactici Pacid-1 was inoculated into 5 mL anaerobic triple mixed medium (BHI+MRS+modified GAM) at an inoculum level of 2%, enterococcus faecalis (beta hemolysis, CICC23658, purchased from China industry microbiological culture Collection center) was used as a positive control, and a blank medium was used as a negative control. All strains were anaerobically cultured in anaerobic triple media at 37℃for 12 h to give activated strains. 2.5. Mu.L of each activated strain was inoculated onto Columbia blood plates (Shanghai family, majia biotechnology Co., ltd.) and 3 replicates were set per group. After anaerobic culture at 37 ℃ for 48 h, observing, forming a completely transparent hemolytic ring with obvious limit around the colony of the positive control strain, which is beta hemolysis; the medium surrounding the Pediococcus acidilactici Pacid-1 colonies was unchanged and was gamma-hemolyzed, i.e., not hemolyzed.
Example 4 antibiotic sensitivity test of Pediococcus acidilactici Pacid-1
According to the requirement of antibiotic sensitivity test in the third section of "microecological live bacteria preparation general theory" of Chinese pharmacopoeia (2020 edition), a triple mixed culture medium (BHI+MRS+modified GAM) is used, the sensitivity of the strain to the antibiotic is measured by adopting an agar diffusion paper sheet method, and the sensitivity level of the strain to the antibiotic is judged according to the size of a bacteriostasis zone.
The results show that Pediococcus acidilactici Pacid-1 is sensitive to 11 antibiotics, penicillin, ampicillin, imipenem, ceftriaxone, tetracycline, clindamycin, levofloxacin, nitrofurantoin, high concentration gentamicin, streptomycin, rifampin, and is erythromycin intermediate.
EXAMPLE 5 Pediococcus acidilactici Pacid-1 Artificial gastric juice resistance test
Strain activation and culture: pediococcus acidilactici is treatedPediococcus acidilactici) Pacid-1 was inoculated in 2% inoculum size to 5 mL anaerobic binary medium and cultured to late logarithmic growth phase.
Treatment of strains with gastric juice: taking a bacterial liquid cultured by 1 mL, centrifuging at 5000 rpm multiplied by 5 min, discarding the supernatant, and re-suspending the bacterial liquid by 1 mL anaerobic and resazurin PBS. 0.1 ml bacterial suspension is supplemented with 0.9 ml anaerobic and resazurin-free PBS as a control group, and 0.1 ml bacterial suspension is supplemented with 0.9 ml artificial gastric juice as an experimental group. Mixing, and anaerobic standing at 37deg.C. The artificial gastric juice group is incubated for 6 h, 0.1 ml bacteria liquid is sucked up and diluted to a proper gradient, and 25 mul of the bacteria liquid is taken out in a three-mixed solid culture medium. 37. Anaerobic culture at C until single colony grows out and counting.
And (3) data processing: strain viability = number of viable bacteria of experimental group/number of viable bacteria of control group x 100%. The results are shown in Table 1 and FIG. 2, and Pediococcus acidilactici @Pediococcus acidilactici) Pacid-1 has better tolerance to artificial gastric juice.
TABLE 1 Pediococcus acidilactici @Pediococcus acidilactici) Survival rate of Pacid-1 after gastric juice treatment
Group of Control group Gastric juice group with pH3.0
Viable count (CFU/mL) （1.07±0.07）×10 9 （8.73±0.01）×10 8
Survival (%) N/A 81.9%
Example 6 antibacterial ability of Pediococcus acidilactici Pacid-1 against pathogenic bacteria
4 common pathogenic bacteria which can cause diarrhea are selected for bacteriostasis capability detection, and pathogenic strain source information is shown in table 2.
TABLE 2 pathogenic bead Source information
Strain name Strain deposit number Strain preservation unit
Pseudomonas aeruginosa CMCC(B)10104 China Institute for food and drug control
Shigella dysenteriae CMCC(B)51252 China Institute for food and drug control
Yersinia enterocolitica CMCC(B)52204 China Institute for food and drug control
Clostridium difficile CICC 22951 China industry microbiological culture Collection center
Preparation of Pediococcus acidilactici Pacid-1 fermentation liquor: after the Pediococcus acidilactici Pacid-1 is activated, the Pediococcus acidilactici Pacid-1 is inoculated into an anaerobic triple mixed culture medium (BHI+MRS+modified GAM) according to an inoculum size of 2 percent, and is subjected to anaerobic culture at 37 ℃ for 48 h, so as to obtain a fermentation broth.
Preparation and coating of pathogenic bacteria: pseudomonas aeruginosa, shigella dysenteriae and Yersinia enterocolitica are aerobic bacteria, and after being activated by TSB broth culture medium, are diluted 50 times in TSB broth culture medium to reach proper concentration, and are coated on TSA solid culture medium by 0.2. 0.2 mL diluted bacteria liquid. Clostridium difficile is anaerobic bacteria, and after activation by a triple mixed culture medium, the clostridium difficile is diluted 50 times to reach a proper concentration by the triple mixed culture medium, 200 mu L of the clostridium difficile is coated on an anaerobic GAM solid culture medium (5% horse serum is added, beijing Soy Bao technology Co., ltd., S9050), 3 oxford cups are placed in each flat plate, 200 mu L of fermentation broth to be detected is added into the oxford cups, clostridium difficile is cultured under anaerobic conditions, and other pathogenic bacteria are cultured under aerobic conditions. After incubation at 37℃for 24 h, the diameter of the zone of inhibition was measured.
As shown in FIG. 3, pediococcus acidilactici Pacid-1 has inhibitory activity against Pseudomonas aeruginosa, shigella dysenteriae, yersinia enterocolitica and Clostridium difficile.
Example 7 in vitro Barrier repair test of Pediococcus acidilactici Pacid-1
Caco-2 cells (North Nature, biotechnology Co., ltd., BNCC. No.: 350769) were inoculated: caco-2 cells were digested with pancreatin cell digests pre-warmed at 37℃and resuspended in DMEM medium (Gibco, C11995500 BT) containing 10% FBS (Gibco, 16000-044) and 1% PS (Gibco, 15140-122) at 1.1X10 5 Seed Density of individual cells/well Caco-2 cells were seeded in 24 well Transwell (permeable cell culture Chamber), 5% CO 2 The culture was allowed to stand in an incubator at 37℃for 21 days.
Strain culture: pediococcus acidilactici is treatedPediococcus acidilactici) Pacid-1, lactobacillus rhamnosus @Lactobacillus rhamnosus) GG (LGG, CICC 6141, china center for type culture Collection of Industrial microorganisms) is inoculated into 5 mL second mixed culture medium (related reagent is deoxidized in advance) respectively in an inoculum size of 5 percent, and an electric heating constant temperature incubator at 37 ℃ is used for anaerobic culture for 24 h. Once subcultured, anaerobic culture 8 h. Taking 1 mL bacterial liquid, and centrifuging at 12000 rpm/min for 3 min. Strains were grown to 1X 10 with DMEM medium containing 10% FBS 7 CFU/mL for use.
The Pediococcus acidilactici is exploredPediococcus acidilactici) Effect of Pacid-1 on intestinal epithelial barrier function in Caco-2 cell model: the experiment is divided into 4 groups, namely a normal control group, a model group, a positive control LGG group and a pediococcus acidilactici Pacid-1 group.
Intestinal epithelial barrier function detection: a barrier injury model was constructed using the inflammatory factors IFN-gamma (PeproTech, AF-300-02) and TNF-alpha (PeproTech, 300-01A). After 21 days of Caco-2 cell culture, after the cells differentiated to form compact monolayer cells, the old culture medium of the lower chamber was sucked off, 800. Mu.L of DMEM medium was added to the lower chamber of the normal control group, and 800. Mu.L of IFN-gamma solution was added to the lower chamber of the model group, the positive control LGG group and the Pediococcus acidilactici Pacid-1 group, respectively. Placing in a 5% carbon dioxide incubator, standing at 37deg.C for culturing 22 h, sucking the solution in the upper chamber and lower chamber, adding 200 μl DMEM medium into the upper chamber of the normal control group, and adding 800 μl DMEM medium into the lower chamber; 200. Mu.L of DMEM medium is added to the upper chamber of the model group, and 800. Mu.L of TNF-alpha solution is added to the lower chamber; in the positive control LGG group, 200 mu L of LGG bacteria solution is added into the upper chamber, and 800 mu L of TNF-alpha solution is added into the lower chamber; 200 mu L of Pediococcus acidilactici Pacid-1 bacteria solution is added into the upper chamber of the Pediococcus acidilactici Pacid-1 group; 800. Mu.L of TNF-. Alpha.solution was added to the lower chamber. After 5% carbon dioxide incubator and stationary culture at 37℃for 5 h, the individual cell monolayer transmembrane resistance (TEER) values were measured.
The results are shown in FIG. 4: LGG can significantly increase TEER values compared to model group, indicating significant repair of cell barrier damage. Likewise, pediococcus acidilactici Pacid-1 also significantly increased TEER values compared to the model group. The result shows that the Pediococcus acidilactici Pacid-1 can effectively relieve barrier dysfunction caused by inflammatory factors (such as IFN-gamma and TNF-alpha).
Example 8 in vitro cell inflammation inhibition assay of Pediococcus acidilactici Pacid-1
THP-1 cell polarization: RPMI-1640 (Thermo Fisher, C11875500 BT) medium containing 10% (v/v) FBS and a final concentration of 100 ng/mL PMA (phorbol 12-tetradecanoate 13-acetate, sigma-Aldrich Company, P1585) was used at 1X 10 5 Seed Density of individual cells/well THP-1 cells were seeded in 96 well plates and placed in 5% CO 2 Incubator, incubation at 37℃for 24 h polarizes into mature macrophages.
Strain culture: pediococcus acidilactici Pacid-1 bacterial liquid 200 mu L to 5 mL mixed culture medium (BHI+MRS) is inoculated from bacterial storage, and the bacterial liquid is subjected to anaerobic culture in a 37 ℃ electric heating constant temperature incubator for 24 h. After one transfer, anaerobic culture 8 h. Taking 1 mL bacterial liquid, and centrifuging at 5000 rpm/min for 15 min. Diluted to 2X 10 with RPMI-1640 medium containing 10% (v/v) FBS 6 CFU/mL was ready for use.
Effect of Pediococcus acidilactici Pacid-1 on the expression of TNF- α and IL-6 by THP-1 cells: THP-1 maturationAfter the cells are cultured, the normal control group is replaced with RPMI-1640 medium containing 10% (v/v) FBS; the model group, the positive control dexamethasone group and the Pacid-1 test group were each subjected to modeling of inflammatory macrophages by exchanging RPMI-1640 medium containing 10% (v/v) FBS, 100.sup. 100 ng/mL LPS (Sigma-Aldrich Company, L3024) and 20.sup. 20 ng/mL IFN-. Gamma.PeproTech, AF-300-02. Each group was exposed to 5% CO 2 In an incubator, 24. 24 h was cultured at 37 ℃. The medium was aspirated, and 100. Mu.L of RPMI-1640 medium containing 10% (v/v) FBS was added to the normal control and model groups, respectively; the positive control group was supplemented with 100. Mu.L of RPMI-1640 medium containing 10% FBS and dexamethasone (Sigma-Aldrich Company, D4902-25) at a final concentration of 25. Mu.g/mL; pacid-1 test group 100. Mu.L of previously prepared Pediococcus acidilactici Pacid-1 bacteria solution was added. Placed in 5% CO 2 After culturing at 37℃in an incubator 24H, 80. Mu.L of the cell culture broth was aspirated, respectively, at 4℃at 5000 rpm/min, centrifuged for 15 min, and the supernatant was collected, and the TNF-alpha content (E-EL-H0109 c, wuhan Irite Biotechnology Co., ltd.) was detected using a Human TNF-alpha (Tumor Necrosis Factor Alpha) ELISA kit, and the IL-6 content was detected using a Human IL-6 (Interlukin 6) ELISA kit (Wuhan Irite Biotechnology Co., ltd., E-EL-H6156).
Test results: as shown in fig. 5, the expression of TNF- α and IL-6 was significantly higher in the model control cells than in the normal control (P < 0.01); positive controls dexamethasone and pediococcus acidilactici Pacid-1 both can significantly inhibit the expression (P < 0.01) of pro-inflammatory factors IL-6 (figure 5A) and TNF-alpha (figure 5B) in THP-1 cells, indicating that pediococcus acidilactici Pacid-1 has very obvious anti-inflammatory effect in vitro.
EXAMPLE 9 therapeutic Effect of Pediococcus acidilactici Pacid-1 on 5-fluorouracil (5-FU) diarrhea mice
And (3) preparing a freeze-drying protective agent:
and (3) solution A: sucrose 8 g, trehalose 8 g, purified water 44 g;
and (2) liquid B: sodium glutamate 2 g, arginine hydrochloride 2 g, purified water 16 g; sterilizing at 115 deg.C for 20 min.
And C, liquid: vitamin C sodium 4 g, purified water 16 g. Filtering and sterilizing for standby.
When in use, the components are mixed according to the volume ratio A, B and C=6:2:2.
Preparing bacterial powder: inoculating the preserved pediococcus acidilactici Pacid-1 and pediococcus acidilactici Pacid-2 into a bacterial powder preparation culture medium according to 10% of inoculum size, and performing anaerobic culture at 37 ℃ and 90 rpm for 14-20 hours to obtain a first-stage seed solution (OD) 600 The value is not less than 2.0). Then transferring the strain to a bacterial powder preparation culture medium according to the inoculation amount of 3.0%, and performing anaerobic culture at 37 ℃ and 90 rpm for 5-10 hours to obtain a secondary seed solution (OD) 600 The value is not less than 2.0). Pumping the secondary seed liquid into a fermentation tank by a peristaltic pump according to the inoculation amount of 1.5%, setting fermentation parameters (37 ℃, pH of 5.1, 100 rpm and 0.06 MPa), and fermenting and culturing. Fermentation broth OD 600 The value is not less than 1.8 or OD 600 Stopping fermentation when the value is increased to be less than or equal to 0.1, setting the fermentation temperature to be 20 ℃, and centrifugally collecting thalli. Adding a freeze-drying protective agent according to the weight ratio of the bacterial mud to the freeze-drying protective agent of 1:1-1:2, and uniformly mixing to emulsify the bacterial mud. And (3) putting the emulsified bacterial suspension into a plate layer of a freeze dryer cooled to the temperature of minus 40 ℃ for freeze-drying, taking out bacterial cakes after the freeze-drying procedure is finished, and crushing to obtain bacterial powder. Pediococcus acidilactici Pacid-1 and Pediococcus acidilactici Pacid-2 were formulated to 5X 10 with physiological saline prior to animal administration 9 CFU/mL of bacterial suspension.
Test animals: 25 SPF-class male Balb/c mice weighing 18-22 g, purchased from Experimental animal technology Co., ltd., beijing, and fed to SPF-class animal houses.
And (3) test design: the mice were induced with 5-FU (5-fluorouracil, available from Tianjin JinYao pharmaceutical Co., ltd., specification 10. 10 mL/count, 0.25 g/10 mL) solution for chemotherapy-related diarrhea model, and were randomly divided into 5 groups according to initial weights of the mice, namely, a normal control group, a model control group, a positive control loperamide group, a Pacid-1 group and a Pacid-2 group, each group being 5.
The overall test period was 9 days and was noted as D1-D9. D3, 5-FU single molding treatment was performed, and 5-FU single molding treatment was performed in the other groups except normal control group to which physiological saline was injected, and molding amounts were administered by weight (350 mg/kg).
All groups of administration modes are lavage, normal control group and model control group lavage freeze-drying protective agent, and the method is continuousGastric lavage for 5 days (D1-D5); the positive control group was continuously gavaged with loperamide (purchased from the western amprensen pharmaceutical limited company, LFJ 8684) for 9 days (D1-D9, 20 mg/kg) on a weight basis; pacid-1 group and Pacid-2 group were intragastric 1X 10 for 5 consecutive days (D1-D5) 9 CFU/bacterial suspension alone. After the end of D5 administration, observations were continued for 4 days. The specific test groups and dosing regimens are shown in table 3.
Table 3 experimental grouping and dosing regimen of pediococcus acidilactici for treating 5-FU diarrhea mice
Group of Quantity of Molding agent Amount of modeling agent Test article Administration volume Dosage for administration Days of administration
Normal control group 5 Physiological saline / Freeze-drying protective agent 0.2 mL/only / 5 d
Model control group 5 5-FU 350mg/kg Freeze-drying protective agent 0.2 mL/only / 5 d
Loperamide group 5 5-FU 350mg/kg Loperamide 10 mL/kg 20 mg/kg 9 d
Pacid-1 group 5 5-FU 350mg/kg Pacid-1 0.2 mL/only 1×10 9 CFU/only 5 d
Pacid-2 group 5 5-FU 350mg/kg Pacid-2 0.2 mL/only 1×10 9 CFU/only 5 d
Note that: 5-FU 5-fluorouracil; CFU colony forming unit colony forming units; d, tiantian
Diarrhea observations and scoring: mice were placed in 1 mouse cage with clean filter paper placed in each cage. Hard feces, normally considered 0 minutes; mild, slightly wet or soft stool was considered 1 minute; moderately wet feces, fecal and anal Zhou Bujie are considered as 2 minutes; severe, thin stool and severe anus Zhou Bujie were considered 3 minutes. During the experimental period, the mouse feces were observed and scored daily. Total diarrhea score was the sum of daily diarrhea scores.
Test results: as shown in fig. 6, pacid-1 had a significant improvement in diarrhea caused by 5-FU compared to the model control group (fig. 6A), D8 diarrhea score was significantly reduced (P < 0.01) compared to the model control group (fig. 6B), total diarrhea score was significantly reduced (P < 0.01) (fig. 6C), and Pacid-2 did not see significant differences compared to the model control group.
The results show that the Pediococcus acidilactici Pacid-1 disclosed by the invention can significantly improve diarrhea caused by the chemotherapeutic drug 5-FU, and the diarrhea improving effect is better than that of Pacid-2.
The above description is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto. Any person skilled in the art, within the scope of the present disclosure, may apply to the present invention, and any equivalent or modified embodiments thereof are included in the scope of the present invention.

Claims (10)

1. Pediococcus acidilacticiPediococcus acidilactici) The strain is Pediococcus acidilactici Pacid-1 with the preservation number of CCTCC NO: M20222030.
2. Pediococcus acidilactici according to claim 1Pediococcus acidilactici) A strain, characterized in that the 16S rDNA sequence of the strain is shown as SEQ ID NO.1, andthe Pediococcus acidilactici has genes encoding the following proteases:
1) An enzyme related to acetic acid production with the amino acid sequence shown as SEQ ID NO.2
2) An enzyme related to acetic acid production with the amino acid sequence shown as SEQ ID NO.3
3) An enzyme related to acetic acid production with the amino acid sequence shown as SEQ ID NO.4
4) An enzyme related to acetic acid production with the amino acid sequence shown as SEQ ID NO.5
5) Propionic acid-producing related enzyme with amino acid sequence shown as SEQ ID NO.6
6) The amino acid sequence is shown as SEQ ID NO.7 to produce CAT related enzyme.
3. A pediococcus acidilactici according to any one of claims 1 to 2Pediococcus acidilactici) The method for culturing the strain is characterized in that the pediococcus acidilactici strain is inoculated into a culture medium, and proliferation culture is carried out to obtain the proliferated pediococcus acidilactici strain.
4. The culture method according to claim 3, wherein the culture medium contains 15-20 g of BHI broth powder, 10-15 g of MRS broth powder, and 12-17 g of modified GAM broth powder per 1-L of distilled water.
5. A food, health food or pharmaceutical composition comprising the Pediococcus acidilactici of any one of claims 1 to 2 as an active ingredientPediococcus acidilactici) A strain or a strain comprising Pediococcus acidilactici obtained by the cultivation method according to claim 3 or 4Pediococcus acidilactici) Strains.
6. Pediococcus acidilactici as described in any one of claims 1 to 2Pediococcus acidilactici) Or the use of a composition according to claim 5 for the preparation of a product for improving intestinal health.
7. The use according to claim 6, wherein the intestinal health condition is intestinal inflammation and/or intestinal barrier damage and/or intestinal pathogen infection.
8. The use according to claim 6, wherein the intestinal health condition is diarrhea caused by a chemotherapeutic agent.
9. The use according to claim 7, wherein the enteropathogenic bacteria are selected from any one or a combination of the following: pseudomonas aeruginosa, shigella dysenteriae, yersinia enterocolitica and Clostridium difficile.
10. The use according to claim 8, wherein the chemotherapeutic agent is selected from one or a combination of the following: 5-fluorouracil, tegafur, 5'-2' -deoxyuridine, capecitabine, tegafur, paclitaxel, docetaxel, vinorelbine, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, ifosfamide, melphalan, carmustine, irinotecan.
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